1 Introduction

Use of nano-scale materials (1–100 nm) has been widespread especially in the last half-century and grew rapidly through all fields involving food as well as various industrial products (e.g., cosmetics, toothpaste, paints, electronics, and pharmaceuticals) [34] which lead to daily human exposure. Since they have unknown characteristics and the data on their potential toxicity is still limited [57], their toxic effect including genotoxicity must be studied. Since these materials entered the field of agriculture as nanofertilizers and nanopesticides, the need to study their effect on plants which are the easiest models to study nano-scale material toxicity (where the interaction between both of them is an effective aspect to assess their predictable dangerous side effects) has been paramount.

Nano-scale materials such as nano-SiO2 (n-SiO2) and nano-TiO2 (n-TiO2) can affect early plant development (Tables S1 and S3). Concentration, particle size and structure of nanomaterials are known to affect plant growth differently. Also, these materials can exert genotoxicity by direct and indirect mechanisms [33]. They can penetrate plant cells [26] to interrupt cell division causing chromosomal abnormalities and cell degeneracy, for example in Lens culinaris (lentil) and Allium cepa (onion) [24, 36, 38, 51], and can generate reactive oxygen species (ROS) which cause DNA damage and cell death [37]. Nano-scale materials show high toxicity and are believed to be more toxic than their bulk material [16]. Plant root-tip cells treated by such materials exhibited many types of chromosomal abnormalities, such as breaks, lagging, disturbance, spindle dysfunction, stickiness, fragments, gaps and multipolarity in mitotic and meiotic cells [1, 9, 40].

Nano-SiO2 which is promising for biological applications due to its excellent biocompatibility and large-scale synthetic availability could stimulate plant growth (Table S1) and is believed to enhance it at low to medium (50–800 mg/L, [3] or high (2000–14,000 mg/L, [48] concentrations. However, this may involve a genotoxic effect on the plant cell mitosis and chromosomal abnormalities [50] (Table S2).

Nano-TiO2 has both positive and negative effects on plant growth [8, 22, 62] (Table S3). Therefore, it must be used gingerly until relevant data enable its safe utilization [52]. Its genotoxicity depends on its particle size and crystalline structure [8, 29] (Table S4); Exposure to the n-TiO2 is capable of inducing genotoxicity in the plant systems even at a low concentration (12.5 mg/L) due to the internalization of the particles and the oxidative stress [36]. Bulk materials of TiO2, however, showed higher toxicity on V. faba than its nano-forms [7], which may need further tests.

Broad bean (Vicia faba L.) (Fabaceae) is one of the most important model plants for ecotoxicology studies [39] and also an important crop as a carbohydrate food source. This work was established to test.

  • n-SiO2 and n-TiO2 effects on V. faba growth through seed germination, root and shoot lengths as well as vigor index.

  • the genotoxic effects of n-SiO2 and n-TiO2 on different types of chromosomal aberrations for six homogeneous chromosomes of V. faba root-tip cells as well as mitotic cell division phases.

2 Materials and methods

2.1 Nano-scale materials

Nano-scale silicon dioxide (n-SiO2) and titanium dioxide (n-TiO2) (anatase) were purchased from Nanotech Egypt Co., Egypt. For visualization purpose only, osmium coating was applied to these nano-scale materials and observation was made under a scanning electron microscope (SEM) (SU8000, Hitachi Hitechnologies) at the Center of Advanced Instrumental Analysis, Kyushu University. Both n-SiO2 and n-TiO2 were suspended in double distilled water by sonication for 30 min before use to make concentrations of 25, 50 and 75 mg/L.

2.2 Seed material

Seeds of commercial broad bean (V. faba) variety Sakha 1 were obtained from Food Legumes Research Section, Sakha Agricultural Research Station, Egypt.

2.3 Experimental procedure

Seeds were surface sterilized with 2.5% sodium hypochlorite (NaOCl) for 3 min, and then were rinsed 3–4 times with distilled water followed by immersing in distilled water for 3 h. The seeds were then soaked for 24 h in three different concentrations 25, 50 and 75 mg/L each of n-SiO2 or n-TiO2 solution, or in distilled water as the control (0 mg/L). After treatment, the seeds were thoroughly washed at least three times with distilled water. The randomized complete block design was used in three replications for both materials. Experiments were carried out at the Laboratory of Genetics Department, Faculty of Agriculture, Kafrelsheikh University, Egypt.

2.3.1 Germination, seedling lengths and vigor index

To study the effects of n-SiO2 and n-TiO2 on seed germination, 15 seeds per replicate were allowed to germinate and grow in a 15 cm diameter Petri dish (four dishes/treatment) lined with filter paper (Whatman No. 1) moistened with distilled water. The Petri dishes were placed in a growth room controlled at 25 ± 1 °C and 12:12 h light:dark photocycle. The seeds were considered germinated when the radicle length reached 3 mm. Germination percentage was inspected at the seventh day for the treated and control seeds as described by Al-Mudaris [2] as follows:

$${\text{Germination}}\;{\text{percentage}}\; = \;\left( {{\text{number}}\;{\text{of}}\;{\text{germinated}}\;{\text{seeds/total}}\;{\text{number}}\;{\text{of}}\;{\text{seeds}}} \right) \times 100$$
(1)

Ten seeds per replication were allowed to grow in pots supplied with peat moss after treatments. Three replications per treatment were prepared. Then, root and shoot lengths were measured in centimeters after 14 days as the mean of succeeded germinated seedlings.

The values of germination percentages in addition to root and shoot lengths were used to calculate the vigor index according to the equation of Dahindwal et al. [10] as follows:

$${\text{Vigor}}\;{\text{index}} = \left( {{\text{shoot}}\;{\text{length}} + {\text{root}}\;{\text{length}}} \right) \times {\text{germination}}\;{\text{percentage/}}100$$
(2)

2.3.2 Cytological analysis

Root tips (1.5–2 cm in length) of germinated seeds were cut and fixed in a fresh solution of glacial acetic acid and absolute ethanol at the ratio of 1:3 for 24 h and then stored in 70% ethanol at 4 °C until use. For cytological analysis, roots were boiled in 45% glacial acetic acid to break connections among cells and facilitate mashing of root tips. Then, about 1 mm from excised root tips were stained with 2% aceto-carmine [11] and squashed on a slide to be examined (about 1000 cells per replicate) under a light microscope at × 1000 magnification. Mitotic index was calculated for each replicate as follows:

$${\text{Mitotic}}\;{\text{index}} = \left( {{\text{number}}\;{\text{of}}\;{\text{dividing}}\;{\text{cells/number}}\;{\text{of}}\;{\text{dividing}}\;{\text{and}}\;{\text{nondividing}}\;{\text{cells}}} \right) \times 100$$
(3)

The number of abnormal cells (showing chromosomal aberration) relative to dividing cells was calculated for each treatment:

$${\text{Percentage}}\;{\text{of}}\;{\text{abnormal}}\;{\text{cells}} = \left( {{\text{number}}\;{\text{of}}\;{\text{abnormal}}\;{\text{cells/number}}\;{\text{of}}\;{\text{dividing}}\;{\text{cells}}} \right) \times 100$$
(4)

2.4 Statistical analysis

Data from each treatment with three replicates were analyzed by nonparametric Wilcoxon/Kruskal–Wallis tests and then by posthoc Tukey tests for multiple comparisons using JMP13.2.1. The results were presented as mean ± SD.

3 Results and discussion

The particle sizes of n-SiO2 and n-TiO2 were 119.1 ± 2.8 and 283.6 ± 15.9 nm, respectively (mean ± SD, n = 10 for each) (Fig. 1).

Fig. 1
figure 1

Scanning electron microscope (SEM) images of nano-SiO2 (left) and nano-TiO2 (right)

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3.1 Effects of n-SiO2 and n-TiO2 on germination and plant growth

All three tested concentrations of both materials, n-SiO2 and n-TiO2, did not affect germination percentage (χ2 = 12.13, df = 6, P = 0.0590, Fig. 2). For n-SiO2, similar results were obtained at the concentrations of 90 and 180 mg/L on V. faba [42] and at even higher concentrations (400–4000 mg/L) on Arabidopsis thaliana [31]. On the other hand, n-SiO2 decreased the germination percentage of A. cepa at 540, 810 and 1820 mg/L [51] and L. culinaris by increasing the concentration up to 300 mg/L [24].

Fig. 2
figure 2

Germination percentage and vigor index (mean ± SD) of Vicia faba treated with nano-SiO2 and nano-TiO2. Bars with the same letters did not differ significantly for vigor index. No significant differences in germination percentage among different treatments

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Similar to our result, n-TiO2 treatments did not influence the seed germination of Triticum aestivum (wheat) [18], Hordeum vulgare (barley) [32] and various plant species even at high concentrations (Table S3). By contrast, n-TiO2 decreased germination percentage of Z. mays (at 50 and 100 mg/L, 5 d exposure, [61], Oryza sativa (rice) (at 2000 mg/L, 10 d exposure, [21], Spinacia oleracea (spinach) (at 6000 and 8000 mg/L, 2d exposure, [62] and various other plants when typically exposed to high concentrations or for a long time (Table S3). On the contrary, n-TiO2 at its lower concentration range can enhance germination of various plants except those belong to Poaceae (Table S3). The lack of evidence of germination enhancement by n-TiO2 in the large monocotyledon family, Poaceae, may need further attention and investigation.

As shown in Fig. 3, both nanomaterials did not show any effect on root length (χ2 = 7.49, df = 6, P = 0.28, Fig. 3). By contrast, n-SiO2 moderated impact of salinity on the root length of L. culinaris [44] and Cucurbita pepo [49]. Significant difference was found among different treatments on shoot length (χ2 = 17.08, df = 6, P = 0.009, Fig. 3); Nano-TiO2 shortened shoots at all concentrations studied, whereas n-SiO2 did not affect their length. The latter result with n-SiO2 is in agreement with the results on Oryza sativa (rice) and Zea mays (maize) despite higher concentration (2000 mg/L) [60]. The former (shortened shoot by n-TiO2) is similar to that observed in Mentha piperita [46]. On the contrary, n-TiO2 had no effect at lower concentrations of 5–20 mg/L on T. aestivum [14] or even at higher concentrations of 200–4000 mg/L on Z. mays [6].

Fig. 3
figure 3

Effects of nano-SiO2 and nano-TiO2 on Vicia faba shoot and root lengths (mean ± SD). Bars with the same letters did not differ significantly for shoot length. Root length was not significantly different among treatments

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All n-TiO2 concentrations significantly reduced the vigor index compared to the control, unlike n-SiO2 treatment which had no significant effect at any applied concentrations (χ2 = 17.61, df = 6, P = 0.0073, Fig. 2), reflecting the shoot length decrease by n-TiO2 (Fig. 3). This is in parallel with the negative effect on the vigor index of V. faba using bulk (non-nano) TiO2 [7] but in contrast to the positive effect on that of V. faba using < 10 nm particles [7] and on the vigor index of four out of five plant species studied at 10-20 mg/L or 10–80 mg/L (Nigella sativa, Alyssum homolocarpum, Carum copticum and Salvia mirzayanii, [22], Table S3. Since larger particles have larger surface area or surface energy, they tend to be more unstable and thus to form aggregates of the scale of micron rapidly [17, 19]. In the case of S. oleracea, n-TiO2 shows a positive effect on vigor index but at higher concentrations (250–4000 mg/L) and with less toxic crystal structure, rutile (i.e., oxidative stress is mitigated by antioxidant; [4, 62].

3.2 Effects of nano-SiO2 and nano-TiO2 on mitotic division in root meristem cells

3.2.1 Mitotic index and mitotic phase

Vicia faba root-tip cells showed different mitotic phases. Both materials at any tested concentrations did not affect the mitotic index, compared to the control (χ2 = 9.56, df = 6, P = 0.14). However, the mitotic index at the highest n-TiO2 concentration (75 mg/L) was significantly lower than that at the lowest n-SiO2 concentration (25 mg/L) (Table 1). The positive effect of n-SiO2 at the low concentration is in contrast to the previous results on L. culinaris at the same concentrations [24] and A. cepa at concentrations of 540–1820 mg/L [51]. Although n-TiO2 may have the potential to decrease cell dividing activity with increasing concentration, these results are, overall, in agreement with earlier studies reporting that even at the high concentrations (> 100 mg/L) mitotic activity was not affected by n-TiO2 [7, 20, 32]. In A. thaliana, expression of more genes were down-regulated than up-regulated by n-TiO2 (10–50 nm) at the studied concentration (500 mg/L) [56]. By contrast, slightly more genes were up-regulated than down-regulated by n-TiO2 (< 150 nm) at 100 mg/L [27]. Additionally, n-TiO2 activated antioxidant enzymes (catalase and peroxidase but not superoxide dismutase), amylase and protease at 10–30 mg/L in A. cepa [30], indicating generation of oxidative stress even at its low concentrations.

Table 1 Composition of mitotic phases (%) and mitotic index of Vicia faba root-tip cells treated with three different concentrations of nano-SiO2 and nano-TiO2
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Proportions of cells in the mitotic phases, metaphase (χ2 = 6.96, df = 6, P = 0.325) and telophase (χ2 = 12.26, df = 6, P = 0.0564), were not different from the control and among different concentrations of n-SiO2 and n-TiO2. By contrast, those in the mitotic phases, prophase (χ2 = 14.23, df = 6, P = 0.027) and anaphase (χ2 = 12.64, df = 6, P = 0.0491), were different among treatments. For cells in prophase, there was a difference between different concentrations of n-SiO2; a higher proportion was in this mitotic phase at a lower concentration (Fig. 4). This is associated with the high mitotic index of the cells of the root germinating from the seeds treated by n-SiO2 at this concentration (Table 1). Similar comparison was made in A. cepa treated with n-TiO2 but rather less share of cells was observed in prophase where mitotic index was high [25]. Prophase includes complex processes (chromosome condensation, centrosome movement, mitotic spindle formation, and nucleoli break down) to prepare for cell division. Nano-SiO2 inactivates proteins by binding and changing their 3D structure but causes no synergistic effect [59]. Various proteins that support these processes during prophase may have been arrested and deterred, or alternatively, promoted by nanomaterials, depending on species. For anaphase, after seeds were treated by low concentrations of n-SiO2, root-tip cells were less frequently observed in this mitotic phase than the control (Fig. 4).

Fig. 4
figure 4

Composition (mean ± SD) among different mitotic phases of Vicia faba root-tip cells treated with nano-SiO2 and nano-TiO2. Bars with the same letters did not differ significantly

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3.2.2 Chromosomal abnormalities

Both n-SiO2 and n-TiO2 increased total chromosomal abnormalities through mitotic phases but in a different way (χ2 = 15.93, df = 6, P = 0.014). Nano-SiO2 escalated chromosomal aberration with increasing concentration (Fig. 5). By contrast, all n-TiO2 concentrations showed the same ability for inducing chromosomal aberration even at the lowest concentration (Fig. 5). This illustrates that n-SiO2 had less toxic effect than n-TiO2 on V. faba root-tip cells particularly at 25 mg/L. The dose-dependent induction of total chromosomal aberrations by n-SiO2 in our study contrasts to the increased mitotic index on L. culinaris at 200 and 300 mg/L [24] and on A. cepa at the concentration of 810 mg/L [51]. On the other hand, application of n-TiO2 increased aberration frequency in V. faba root-tip cells. In this respect, the previous studies on Z. mays [7], A. cepa [20, 36] and Vicia narbonensis [6] recorded a high frequency of chromosomal aberrations as a result of n-TiO2 treatments. Nano-SiO2 and n-TiO2 could disturb both chromosome structure and spindle fibers during mitosis. Different types of chromosomal aberrations were observed in the tested materials, such as stickiness (Fig. 6a, b, e), c-metaphase (Fig. 6d), disturbance (Fig. 6c, f), lagging chromosomes (Fig. 6f, g), fragments (Fig. 6e), bridges (Fig. 6h, i) and breaks (Fig. 6b, f). There were neither chromosomal gaps nor multipolarity observed during mitosis nor micronucleus during interphase.

Fig. 5
figure 5

Frequency of abnormal mitotic cells (relative to total dividing cells) (mean ± SD) of Vicia faba root-tip cells treated with nano-SiO2 and nano-TiO2. Bars with the same letters did not differ significantly

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Fig. 6
figure 6

Types of chromosomal abnormalities observed in Vicia faba root-tip cells and induced by application of nano-SiO2 and nano-TiO2: a sticky prophase, b sticky metaphase with break, c disturbed metaphase, d c-metaphase, e fragment in sticky metaphase, f disturbed anaphase with breaks and laggards, g forwarded laggard in anaphase, h bridge in anaphase and i bridge in telophase

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Chromosomal bridges and breaks occurred differently among treatments; n-TiO2 had an ability to break chromosomes (χ2 = 14.41, df = 6, P = 0.025, Fig. 7), which may lead to losing genetic material [45]. Frequency of chromosomal breaks were not different from the control at all n-SiO2 concentrations, whereas it was higher than the control at 50 mg/L n-TiO2 (Fig. 7). Nano-SiO2 promoted chromosome bridges particularly at 50 mg/L, whereas n-TiO2 did not affect their occurrence (χ2 = 12.69, df = 6, P = 0.048, Fig. 7). The higher occurrence of metaphase and anaphase chromosomal aberrations such as bridges and breaks may be due to physical interaction to interrupt chromatin structure or chemical interaction with nuclear protein and mitotic spindle fibers [12, 33]. By contrast, frequency of the following types of aberrations did not differ significantly from those in the control and among different treatments; disturbance (χ2 = 12.36, df = 6, P = 0.054), laggard (χ2 = 6.68, df = 6, P = 0.35), stickiness (χ2 = 5.71, df = 6, P = 0.45), c-metaphase (χ2 = 9.42, df = 6, P = 0.15) and fragment (χ2 = 9.84, df = 6, P = 0.13) (Fig. 7).

Fig. 7
figure 7

Frequency of mitotic abnormalities (mean ± SD) in Vicia faba root-tip cells treated with nano-SiO2 and nano-TiO2. Bars with the same letters did not differ significantly. NS, non-significant

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To summarize, mitosis in root-tip cells and plant development of broad beans incurred larger damage by n-TiO2 than by n-SiO2 even at its low concentrations (i.e., higher chromosomal aberration frequency, higher chromosomal break frequency, and shorter shoots). Mitotic abnormality was induced dose-dependently by n-SiO2 but not reflected in plant development. Toxicity of nano-scale materials in the early stages of plant growth is likely to be due to the following factors: (1) chemical and physical properties that influence the release of ions or the aggregation of particles in more stable forms and (2) the size and shape of the particles, which determine the specific surface area of these materials [5, 35, 60]. The penetration of metal ions into the cell causes cross-link in the DNA, sister chromatid exchange, and mutations [43]. Nanomaterials also cause clogging of pores and barriers in the apoplastic stream and this reduces photosynthesis, generates ROS and damages DNA structures [13, 55, 58]. In our study, although both nanomaterials induced chromosomal aberrations in the root-tip cells, they did not affect mitotic index, seed germination and root elongation, whereas n-TiO2 inhibited shoot elongation. This may be partly due to the large particle sizes (> 100 nm) which could prevent the nanomaterials’ penetration of the seed coat but might allow the nanomaterials to penetrate the radicle (root) cell wall to be transported to the shoot tissue [32].